As a light switch can provide instant illumination (ON) or sudden darkness (OFF), so, too, can gene expression systems provide a gene product at will. Such regulated gene expression systems, wherein specific promoters and regulatory elements that play the role of a light switch are functionally-linked to a gene of interest, are important tools that facilitate gene function studies and enable exogenous control in gene therapies. While endogenous gene expression is tightly regulated, being turned ON and OFF according to developmental state, extracellular and intracellular cues and environmental signals, exogenous gene expression has most often been accomplished by constitutive expression (the light is always ON) wherein transcription is controlled from an unregulated, strong promoter sequence. However, such expression can result in over-expression, temporal mis-expression and cell lethality; hampering gene function studies and gene therapy interventions, and even rendering them ineffective. Although today, regulated (inducible) gene expression systems are readily available, they often suffer from high basal expression (even OFF, the light still flickers), low protein synthesis (the light bulb is dimly illuminated) and over-riding cellular circuits (factors) that interfere with the intended molecular switch. In many cases, the agent that flips the switch itself often exerts pleiotropic effects, becomes ineffective over time, or the safety of its administration to an organism or cell is unknown.
To obviate these and other difficulties, inducible expression systems have been developed. Such systems subjugate the gene(s) of interest under the control of an “inducible” promoter—that is, a promoter to which transcription factors, etc., can be made to bind at will, using exogenous factors such as metals, temperature, hormones, or other polypeptides provided in trans. However, most inducible systems are not completely “turned off” in the absence of the inducer—low levels of basal expression are often observed. Such basal expression reduces the advantages of an inducible expression when the gene to be induced is lethal to the target cell (Ausubel et al., 1987). Desirable characteristics of inducible gene expression systems are presented in Table 1.
TABLE 1Desirable characteristics of inducible expression systems1ElementDesirable characteristicsBasal (uninduced)Very low to none of operably-linked sequenceexpressionInducibilityHigh inducibility and specificityHigh inducer specificityHigh response dynamic range with respect to inducerconcentrationsQuick response to inducerRapid switch-off after removing inducerInducingNon-toxic to preferably the organism, or at least targetagenttissues and cellsInducer exerts no other physiological effectsInducer is absent from target tissues and cellsInducer is appropriate for use in cell culture, animalmodels and humans1Adapted from (Zuo and Chua, 2000).
Common eukaryotic (or adaptable to eukarayotes) inducible expression systems, their characteristics, advantages and disadvantages are presented in Table 2.
TABLE 2Common eukaryotic inducible gene expression systems2Induction ratio(foldSysteminduction)AdvantagesDisadvantagesReferencesHeat shock 4–101. Inducing agent readily available and1. Many pleiotropic effects.(Kothary et al.,inexpensive,2. Limited utility in mammalian1989)2. Very fast induction kinetics (1 h).systems.3. First reported to likely have lowleakiness; however, due to low induction,more likely that leakiness is high.Heavy metal 5–101. Fast induction kinetics (16 h).1. Many pleiotropic effects.(Filmus et al.,ions2. High leakiness.1992)Interferon2–501. Low leakiness.1. Slow induction kinetics (3–4 days).(Kuhn et al.,2. Function dependent on cell type.1995)3. Induction causes many pleiotropiceffects.FK506 dimer  0–1.51. Very fast kinetics (16 h).1. Limited usefulness; mostly in vitro(Belshaw et al.,2. Very low leakiness.to activate endogenous signaling1996)3. Few pleiotropic effects.pathways.Steroid 0–2001. Fast induction kinetics (24 h).1. High leakiness,(Kuo et al.,hormone2. Many pleiotropic effects.1994)3. In vivo utility unknown.Gal4-Er 0–1001. Very fast induction kinetics (1–2 h).1. In vivo utility unknown.(Braselmann et2. Low leakiness.al., 1993)3. No pleiotropic effects.Progesterone10–501. Fast induction kinetics1. RU486 is unspecific for(Wang et al.,antagonist/(10 h, in vitro).progesterone receptors.1994)RU4862. Low leakiness2. Not an ideal induction agent,3. Few pleiotropic effectsespecially for potentially-pregnant/in most subjects.pregnant subjects.4. Receptor is endogenous to3. Lower induction.many subjects4. Receptors are not expressed by5. Inducer is readily availableevery cell types; limited tissueand commonly useddistribution.Mutantunknown1. Low leakiness.1. Slow induction kinetics (3–4 days).(Zhang et al.,estrogen2. Few pleiotropic effects in1996)receptormost subjects.Ecdysone  0–1041. Ecdysone is not produced1. Low polypeptide production.(No et al.,by most sublects.2. Effects of ecdysone (or synthetic1996)2. Fast induction kineticsanalogues) on mammalian physiology over(20 h).time are unknown.3. Very low leakiness,4. No pleiotropic effects,Tetracycline1000–105  1. Tetracycline is not endogenous 1. Higher basal expression of(Gossen et al.,to subjectsoperably-linked subcloned nucleic acid1995)2. Tetracycline is readily available and2. Tetracycline is continuouslycommonly usedadministered to a cell or subject (in the3. Low leakinessversion that is OFF in the presence of4. Induction occurs upon thetetracycline)introduction of tetracycline—3. tetracycline has undesirable sidethus eliminating continuouseffects, such as squelching non-tetracycline administrationspecifically gene expression.(in the version that is OFF4. In cultured cells, tetracycline isin the absence of tetracycline).difficult to wash out and thus itis difficult to synchronizepolypeptide production5. Unreliable; does not always work.lac repressor-  5–10001. Fast induction kinetics (12–24 h)1. Induced expression is commonly(Baim et al.,based2. IPTG inducing agent is not producedlimited to only 40 to 50-fold1991)in subjects.2. IPTG can exert cytotoxic effects.3. IPTG is easily available.3. Unreliable; moderate success.4. High leakiness.Heat Shock
The initial constructs took advantage of the D. melanogaster hsp70 promoter that relied on the highly conserved endogenous heat shock transcription factors for binding upon heat shock. Upon transfer from physiological to elevated temperatures in transient transfections, induction was rapid, subsided after cells were returned to physiological temperatures, and basal expression was low (Schweinfest et al., 1988). An in vivo vector was also constructed for transgenic mice, although induction was non-uniform throughout analyzed embryos, most likely due to uneven access of the induction agent (heat) to the different parts of the animal, as well as tissue differences in the ability to respond to heat shock stress (Kothary et al., 1989). However, heat shock inducible systems are best suited for short-term studies, since subjects will pleiotropically respond to heat shock (Gingrich and Roder, 1998).
Heavy Metal Ions
These systems exploit the regulatory elements of metallothionein genes; the most useful heavy metal ions being zinc (Zn2+) and cadmium (Cd2+). These systems comprise metal responsive elements within metallothionein gene promoters that are bound by metal-activated factors. Because metallothionein promoters are also recognized by constitutively expressed transcription factors and are also sensitive to glucocorticoids, progesterone, interleukin-5 and interferon, basal expression levels are high; consequently, induction is often low (Gingrich and Roder, 1998). To improve their usefulness, these promoters are combined with other inducible elements, often with other transcription factor-sensitive regions deleted, resulting in synergistic induction (Filmus et al., 1992). For example, melding of a metal-sensitive promoter region with that of a dexamethasone region results in 65-fold induction, while either the metal ion or dexamethasone gave less than 10% of those levels by themselves. However, in addition to high basal expression levels, heavy metals are toxic over time to mammalian subjects, limiting these system's usefulness.
Interferon
Interferon is produced in response to viral infection, double stranded RNA, mycoplasma, bacteria, endotoxin and antigens (David, 1995). When interferon cell surface receptors are liganded, signaling pathways are activated that result in the binding of transcription factors to interferon stimulated response elements in the promoter regions of interferon-sensitive genes. However, because many genes are interferon-sensitive and mammals constitutively express low levels of interferon, using interferon stimulated response elements results in uneven expression, even between genetically identical organisms and exerts pleiotropic effects. Constitutive high levels of interferon may also be adverse to a subject (Gingrich and Roder, 1998).
Specific Induction of Signaling Pathways (e.g., FK506 Dimer)
In this system, controlled gene expression is mediated by activating specific signal transduction pathways. Signaling is induced by a lipid-soluble synthetic ligand that promotes the oligomerization (cross-linking) of cell surface receptors that lack transmembrane and extracellular regions. This signaling then promotes specific transcription factors to bind to their promoter regions, inducing gene expression. The prototype of this system was first performed using a dimer of the synthetic ligand FK506, specific for immunophilin FKBP12. NFAT-responsive promoter regions, once bound by NFAT transcription factor, promote gene expression (Spencer et al., 1993). However, while useful for investigating signal transduction pathways, this approach has limited usefulness for specific inducible gene expression, since such transduction pathways usefully affect many other genes in addition to the target (Gingrich and Roder, 1998).
Steroid-based Inducible Expression Systems
Glucocorticoid (Glucocorticoid Responsive Elements)
Glucocorticoid-induced expression systems take advantage of glucocorticoid responsive elements found in various promoters. While some improvements have been made—such as combining glucocorticoid responsive elements with other inducible systems, such as heavy metals, combining with tissue-specific promoters, modified ligands/receptors, etc.—these systems are fraught with high basal expression and the possibility for many pleiotropic effects.
Gal4-Estrogen Receptor
This system uses a fusion protein of yeast Gal4 (DNA binding domain) and mammalian estrogen receptor hormone-binding domain (Braselmann et al., 1993). This system is more selective than most: no mammalian cells express Gal4 and few express estrogen receptors in vitro (although in vivo, many tissues do). Target genes are operably-linked to Gal4-binding sites, preferably artificial ones such that basal expression is low. Upon estrogen administration, the fusion receptor binds estrogen and then binds to the Gal4 binding sites, initiating transcription. Because estrogen receptors exhibit cell-type specificity due to two monacidic transactivation functions (TAF-1 and -2), the Herpes simplex virus virion protein 16 acidic activation domain (VP16; a strong activator (Triezenberg et al., 1988)) was added to the fusion protein, thus increasing transcription. Even though the VP16 fusion results in good (100-fold) induction; the system is unattractive in that estrogen has many non-specific effects.
Mutant Progestrone Receptor
In this system, a 42 amino acid C-terminal deletion mutant of the human progesterone receptor that poorly binds progesterone but does bind RU486 (Mifepristone) and other progesterone receptor antagonists, is exploited. Fused with the VP16 activation domain, this chimeric protein can induce expression from those sequences operably-linked to Gal4 DNA binding domains. While exhibiting fast induction, only modest expression is observed. While the RU486 doses sufficient for induction are modest and less than that required for anti-progesterone activity and in vivo efficacy (Wang et al., 1994), the long-term effects of chronic RU486 administration at these doses causes concern. Induction is high, basal expression is moderate and the system is suitable for gene therapy because RU-486 is a human pharmaceutical. The main disadvantage lies in the use of a progesterone receptor mutant that activates many cellular genes usually regulated by progesterone receptors.
Mutant Estrogen Receptor
These systems are based on mutant estrogen receptors that bind the anti-estrogen pharmaceuticals tamoxifen or 4-hydroxytamoxifen, but not estrogen (17β-estradiol) (Zhang et al., 1996). Fusing the mutant estrogen receptor to Cre recombinase and assaying the ability to mediate lox-based recombination demonstrated efficacy of this system. High tamoxifen doses induced recombination, although even slightly higher doses were cytotoxic. Over time, the basal activity of this chimeric protein increased; however, fusing the mutant estrogen receptor to the fusion protein termini reduced basal activity levels. However, this system has not been useful for inducing expression from an operably-linked polynucleotide to estrogen receptor DNA binding domains. These Cre-estrogen receptor chimeras are useful for producing knockout and transgenic mice but are not used for the production of recombinant polypeptides in cultured cells.
Ecdysone
Ecdysone, an insect steroid, triggers morphological changes in D. melanogaster. To mediate ecdysone activity, ecdysone receptors form heterodimers with an insect homolog of vertebrate retinoid X receptor (RXR), ultraspiricle. Ecdysone's effects are exerted even if ultraspiricle is replaced with the human ultraspiricle homolog, RXR, in mammalian cells. An optimized ecdysone promoter is operably linked to a polynucleotide of interest, and a modified ecdysone receptor (fused with VP16) is expressed. Ecdysone (or muristerone) induction results in liganded ecdysone receptors, which then bind to the promoter, inducing expression. In short-term studies, mice did not exhibit side effects from muristerone administration (No et al., 1996). Generally, induction is very high, basal expression levels are low; however, protein synthesis is also low. Because the system uses a synthetic hormone related to those seen in fruit flies, this system may not be suitable for human gene therapy applications if, for example, muristerone engenders pleiotropic effects.
Prokaryote-based Inducible Systems
E. coli lac Repressor
When bound to lac operator DNA sequences, lac repressor inhibits transcription of operably-linked polynucleotide sequences. When bound to lactose or non-metabolizable isopropyl-β-D-thiogalactopyranoside (IPTG), the lac repressor releases the operator sequences, thus allowing transcription to proceed. Adding lac operator sites near the TATA-box or promoter start sites enables the operably-linked polynucleotides to be regulated by lac repressor in eukaryotes.
Because lac operator/lac repressor complexes also efficiently terminate transcription, lac operator site(s) inserted downstream of an operably-linked promoter can also be used to manipulate gene expression (Deuschle et al., 1990).
In another version, lac repressor is fused to VP16, and operator sites are operably-linked upstream of a minimal promoter. The lac repressor was converted to a mammalian transcriptional activator by adding a nuclear localization signal and VP16. IPTG or lactose thus do not induce expression, but silence it. While induction levels are high, chronic administration of IPTG reduces precise control of gene expression (Labow et al., 1990). The reverse of this system was created, although in to this instance, lac repressor binding to operator sites was found to be heat-sensitive (Baim et al., 1991). Other attempted improvements resulted in various problems (Gingrich and Roder, 1998).
Tetracycline
Currently this system is the most popular for controlling exogenous gene expression in many types of cultured cells. Fusing E. coli tet repressor with VP16 creates a tetracycline-controlled transactivator that in the presence of tetracycline or derivatives, can induce expression from polynucleotides operably linked to tet operators (Gossen et al., 1995); this approach was an improvement on an earlier one that lacked the VP16 fusion, wherein tetracycline is chronically administered to silence expression; removal of tetracycline induces expression. However, sufficient tet repression levels could not be consistently obtained (Gossen and Bujard, 1992). While the system exhibits desirable characteristics, it suffers from the problems of clearing tetracycline and derivatives from cells (Gingrich and Roder, 1998). In general, fold induction is good and the expression level is high.
While many inducible expression systems are available, systems that incorporate and exhibit the largest number of desirable characteristics (Table 1) and overcome the majority of limitations of current systems will give the medical and research fields excellent tools to treat (via gene therapy, for example) and discover basic biological mechanisms in development, cell biology and molecular biology.